Suppressed Dickkopf-3 (DKK3) expression is a hallmark of many human cancers and expression levels are inversely related to tumor virulence (e.g., in prostate cancer and ovarian cancer). Using prostate cancer as an example, over-expression of DKK3 halts proliferation of prostate cancer cells, but the beneficial consequences of DKK3 over-expression in both in vivo and ex vivo models of prostate cancer are likely an artifact of the inadvertent initiation of an ER stress response in cells attempting to process an over-expressed, exogenous, secretory gene product. (Abarzua, et al. 2005 Cancer Res 65(21): 9617-22; Abarzua, et al. 2008 Biochem Biophys Res Commun 375(4): 614-8; Abarzua, et al. 2007 Int J Mol Med 20(1): 37-43.)
The tumor suppressor activity of DKK3 was also reported to be due to its ability to block the translocation of β-catenin to the nucleus by forming an inactive complex composed of a cytoplasmic ˜30 kDa DKK3 gene product and βTrCP. (Lee, et al. 2009 Int J Cancer 124(2): 287-97.) Since it is unlikely that chronic ER stress is the mechanism by which endogenous DKK3 gene products influence normal cell proliferation, the identification of a non-secreted, intracellular version of DKK3 and the discovery of two intracellular events (JNK activation and β-catenin inactivation) that facilitate growth arrest offer an opportunity to define the molecular events mediating the DKK3 tumor suppressor function in the prostate.
Early studies by Dr. Leonard and colleagues discovered that the DKK3 gene locus encodes a second transcript that produces an intracellular membrane associated 29 kDa protein (formally D2p29, now renamed, DKK3b). (Leonard, et al. 2000 J Biol Chem 275(33): 25194-201; Farwell, et al. 1996 J Biol Chem 271(27): 16369-74; Safran, et al. 1996 J Biol Chem 271(27): 16363-8; Farwell, et al. 1993 J Biol Chem 268(7): 5055-62.)
Subsequent studies revealed that this DKK3b gene product originated from a second transcriptional start site located in intron 2. Dkk3b was originally identified in astrocytes as a highly trafficked membrane protein that binds thyroid hormone with high affinity. Analysis of the DKK3 gene locus revealed that DKK3b is encoded by exons 3-8 and that a functional transcriptional start site—with a TATA box—is located in intron 2 (FIG. 1A). Promoter mapping studies narrowed the promoter activity to ˜250 bases upstream of exon 3. Deletion of the TATA box blocked promoter function. ChIP analysis revealed that this promoter was functional in vivo (FIG. 1B).
Importantly, in both the full length DKK3 and truncated DKK3b mRNAs, the only authentic Kozak start site is located at the Met beginning at exon 3, and in vitro translation of the full-length DKK3a mRNA using Kozak context dependent conditions yields a 29 kDa protein. (Leonard, et al. 2000 J Biol Chem 275(33): 25194-201.) Real time PCR analysis of DKK3 locus transcripts revealed that the DKK3a transcript (exons 2-8) accounted for ˜55% of the total DKK3 mRNAs, while the DKK3b transcript (exons 3-8) contributed ˜45% of the total DKK3 mRNAs. In the ΔDKK3 mouse that lacks exons 2, full-length DKK3a transcripts are lost, but the DKK3b mRNA is preserved (FIG. 1C). (Barrantes, et al. 2006 Mol Cell Biol 26(6): 2317-26.) Affinity labeling of DKK3b associated with the cellular membranes of ΔDKK3 mouse brain yielded the anticipated immuno-reactive DKK3b, while a full-length glycosylated DKK3 was not synthesized (FIG. 3D). (Farwell, et al. 1989 J Biol Chem 264(34): 20561-7.) These data demonstrate that the DKK3 locus encodes two functional transcripts; one encoding a secreted glycoprotein identified as DKK3a, and another an intracellular 29 kDa DKK3b protein.
There remains an ongoing need for establishing novel therapeutic approaches to cancer treatment utilizing DKK3b tumor suppressor function, for example, treatment methodologies and pharmaceutical compositions that can arrest the growth of various cancers, such as ovarian and prostate cancers.